REPORT 

Daphnia sp., 48-Hour Acute Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

SParr 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

41206434 

26 April2013 

Page 1 of 41 
Harlan Study Number: 41206434 Report Page2 

CONTENTS 

CONTENTS .................................................................................................................................... 2 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ................................................... .4 

QUALITY ASSURANCE STATEMENT ...................................................................................... 5 

SIGNATURE OF CONTRIBUTING SCIENTISTS ...................................................................... 6 

SUMMARY .................................................................................................................................... ? 

Introduction ............................................................................................................................ 7 

Methods ............................................................................................................................... 7 

Results .................................................................................................................................. 7 

GENERAL INFORMATION ......................................................................................................... 8 

Schedule ................................................................................................................................. 8 

Archiving ........................................................................................... , ................................... 8 

1 INTRODUCTION AND PURPOSE ..................................................................................... 9 

1.1 Guidelines I Regulations .......................................................................................... 9 

2 TEST ITEM ........................................................................................................................... 9 

3 MATERIALS AND METHODS ......................................................................................... 10 

3.1 Definitions and Abbreviations ............................................................................... 10 

3.2 Test Species ........................................................................................................... 10 

3.3 Test Water .............................................................................................................. 10 

3.4 Procedure ............................................................................................................... 11 

3.4.1 Range-finding Test .................................................................................. 11 

3.4.2 Definitive Test ......................................................................................... 11 

4 RESULTS ............................................................................................................................ 14 

4.1 Range-finding Test ................................................................................................ 14 

4.2 Definitive Test ....................................................................................................... 14 

4.2.1 Immobilization Data ................................................................................ 14 

4.2.2 Observations on Test Item Solubility ...................................................... 15 

4.2.3 Physico-Chemical Measurements ........................................................... 15 

4.2.4 Verification of Test Concentrations ........................................................ 15 

5 CONCLUSION .................................................................................................................... 16 

6 REFERENCES .................................................................................................................... 17 

TABLES ........................................................................................................................................ 18 

FIGURES ...................................................................................................................................... 20 

APPENDICES ............................................................................................................................... 22 

TABLES ........................................................................................................................................ 34 

FIGURES ...................................................................................................................................... 37 

LIST OF TABLES 

Table 1 

Table 2 

Cumulative Immobilization Data in the Range-finding Test.. .................................... 18 

Cumulative Immobilization Data in the Definitive Test.. ........................................... 19 
Harlan Study Number: 41206434 Report Page 3 

LIST OF FIGURES 

Figure 1 

Figure 2 

Concentration-Response Curve after 24 Hours .......................................................... 20 

Concentration-Response Curve after 48 Hours .......................................................... 21 

LIST OF APPENDICES 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Appendix 5 

Appendix 6 

Reconstituted Water- Elendt M7 Medium ........................................................... 22 

Positive Control ..................................................................................................... 24 

Reconstituted Water .............................................................................................. 25 

Analytical Investigations ....................................................................................... 26 

Physico-Chemical Measurements .......................................................................... 40 

Monitoring Authority Statement ofGLP Compliance ......................................... .41 
Harlan Study Number: 41206434 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Harlan Study Number: 41206434 

Study Title: Daphnia sp., 48-Hour Acute Immobilization Test 

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory 

Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are 

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice 

(revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the 

requirements of Directives 2004/9/EC and 2004/1 0/EC. 

These principles are compatible with Good Laboratory Practice regulations specified by 

regulatory authorities throughout the European Community, the United States (EPA and FDA), 

and Japan (MHL W, MAFF and METI). 

This report fully and accurately reflects the procedures used and data generated. There were no 

circumstances considered to have affected the integrity ofthe study or the validity ofthe data. 

Study Director: SParr 

········~··········· Date: 2 6 APR ?013 
Harlan Study Number: 41206434 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 41206434 

Study Title: : Daphnia sp., 48-Hour Acute Immobilization Test 

The general facilities and activities are inspected at least once a year and the results are reported 

to the relevant responsible person and management. 

Study-related procedures conducted at the test facility were audited and inspected. The details of 

these audits and inspections are given below. 

Reported to the 

Dates and Types of QA Inspections relevant Study 

Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

19 November 2012 General Study Plan N/A 19 November 2012 

Verification 

15 January 2013 Process - based Test Item Preparation 15 January 2013 

10 January 2013 Process - based Test System 10 January 2013 

Preparation 

15 January 2013 Process - based Exposure 15 January 2013 

10 January 2013 Process - based Assessment of 10 January 2013 

Response 

11 January 2013 Process - based Chemical Analysis 11 January 2013 

09 April 2013 Report Audit N/A 09 April 20 13 

The complete study plan (including Study Specific Supplement) was verified for GLP 

compliance at report audit. 

This statement confirms that this report reflects the raw data and the procedures followed. 

Quality Assurance: 

.L~ .... ~:-:.~.~ .. Date: 

2 4 APR 2013 
Harlan Study Number: 41206434 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: DMMullee 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

····~············~·~············Z~13 Date: 
Harlan Study Number: 41206434 Report Page 7 

SUMMARY 

Introduction 

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The 

method followed was designed to be compatible with the OECD Guidelines for Testing of 

Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as 

Method C.2 of Commission Regulation (EC) No. 440/2008. 

Methods 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were 

exposed to an aqueous solution ofthe test item at concentrations of 1.0, 3.2, 10,32 and 100 mg/L 

for 48 hours at a temperature of 20 °C to 21 °C under static test conditions. The numbers of 

immobilized Daphnia were recorded after 24 and 48 hours. 

Results 

The 48-Hour ECso for the test item to Daphnia magna based on nominal test concentrations was 

8.5 mg/L with 95% confidence limits of 6.6 - 11 mg/L. The Lowest Observed Effect 

Concentration was considered to be 10 mg/L. The No Observed Effect Concentration was 

3.2 mg/L. 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be 

near nominal and so the results are based on nominal test concentrations only. 
Harlan Study Number: 41206434 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Archiving 

11 December 2012 

07 February 2013 

Page 8 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), all raw data (paper and electronic) and the final report will be retained in the Harlan 

Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as 

to further retention or disposal. Further retention or return of the data will be chargeable to the 

Sponsor. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Harlan Study Number: 41206434 Report Page9 

1 INTRODUCTION AND PURPOSE 

This report contains a description of the methods used and results obtained during a study to 

investigate the acute toxicity of the test item to Daphnia magna. 

Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, 

and can therefore be considered as an important non-target organism in freshwater ecosystems. 

1.1 Guidelines I Regulations 

This study was designed to be compatible with the procedures indicated by the following 

internationally accepted guidelines and recommendations: 

• OECD Guidelines for Testing of Chemicals (April2004) No 202 "Daphnia sp., Acute 

Immobilisation Test" 

• Method C.2 of Commission Regulation (EC) No. 440/2008 

2 TESTITEM 

The integrity of the supplied data relating to the identity, purity and stability of the test item is 

the responsibility of the Sponsor. 

Identification: 

Description: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 
Harlan Study Number: 41206434 Report Page 10 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Immobilization: 

ECso: 

LOEC (Lowest Observed 

_Effect Concentration): 

NOEC (No Observed _Effect 

Concentration): 

Treatment: 

3.2 Test Species 

Those organisms which are not able to swim within 15 seconds 

after gentle agitation of the test container are considered to be 

immobile. 

The calculated concentration of test item which results in 50% 

immobilization within the exposure period. 

The lowest test concentration at which a significant toxic effect 

on the test organisms is observed within the exposure period. 

The highest test concentration or loading rate at which no 

significant toxic effect on the test organisms is observed within 

the exposure period. 

Comprises test item treatments, control (test water only) and 

solvent control (if applicable). 

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory 

cultures. 

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium 

(see Appendix 1) in a temperature controlled room at approximately 20 °C. The lighting cycle 

was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk 

transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus 

subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction 

was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such 

that the young daphnids produced overnight were less than 24 hours old. These young were 

removed from the cultures and used for testing. The diet and diluent water are considered not to 

contain any contaminant that would affect the integrity or outcome of the study. 

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the 

reference item. Details of the positive control are given in Appendix 2. The positive control was 

conducted between 6 June 2012 and 8 June 2012. 

3.3 Test Water 

Reconstituted water used for both the range-finding and definitive tests is defined in Appendix 3. 
Harlan Study Number: 41206434 Report Page II 

3.4 Procedure 

3.4.1 Range-finding Test 

The test concentrations to be used in the definitive test were determined by a preliminary 

range-finding test. 

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations 

of 0.1 0, 1.0, 10 and 100 mg/L. The test item was dissolved directly in reconstituted water. 

An amount oftest item (1 00 mg) was dissolved in reconstituted water and the volume adjusted to 

1 liter to give the 100 mg/L test concentration. Serial dilutions in reconstituted water were then 

performed from the 100 mg/L test concentration to give the 0.10, 1.0 and 10 mg/L test 

concentrations. 

Each test concentration was inverted several times to ensure adequate mixing and homogeneity. 

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained 

in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and 

8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. 

Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce 

evaporation. After 24 and 48 hours the number of immobilized Daphnia magna were recorded. 

The control group was maintained under identical conditions but not exposed to the test item. 

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to 

determine the stability of the test item under test conditions. All samples were stored at 

approximately -20 °C prior to analysis. Only concentrations within the range to be used for the 

definitive test were analyzed. 

3.4.2 Definitive Test 

Based on the results of the range-finding test the following test concentrations were assigned to 

the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L. 

3.4.2.1 Experimental Preparation 

For the purpose of the definitive test the test item was dissolved directly in reconstituted water. 

An amount oftest item (500 mg) was dissolved in reconstituted water and the volume adjusted to 

5 liters to give the 100 mg/L test concentration. Aliquots (20, 64, 200 and 640 mL) of the 

100 mg/L test concentration were then each separately added to a final volume of 2 liters of 
Harlan Study Number: 41206434 Report Page 12 

reconstituted water to give the further test concentrations of 1.0, 3.2, 10 and 32 mg/L 

respectively. 

Each prepared concentration was inverted several times to ensure adequate mixing and 

homogeneity. 

The concentration and stability of the test item in the test preparations were verified by chemical 

analysis at 0 and 48 hours (see Appendix 4). 

3.4.2.2 Exposure Conditions 

As in the range-finding test 250 mL glass jars containing approximately 200 mL of test 

preparation were used. At the start of the test 5 daphnids were placed in each test and control 

vessel at random, in the test preparations. Four replicate test and control vessels were prepared. 

The test vessels were then covered to reduce evaporation and maintained in a temperature 

controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light and 8 hours darkness with 

20 minute dawn and dusk transition periods with a light intensity ranging from 651 to 669 lux. 

The daphnids were not individually identified, received no food during exposure and the test 

vessels were not aerated. 

The control group was maintained under identical conditions but not exposed to the test item. 

The test preparations were not renewed during the exposure period. Any immobilization or 

adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The 

criterion of effect used was that Daphnia were considered to be immobilized if they were unable 

to swim for approximately 15 seconds after gentle agitation. 

3.4.2.3 Physico-Chemical Measurements 

Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen 

concentrations and pH were recorded at the start and termination of the test. The pH and 

dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter 

whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. 

3.4.2.4 Verification of Test Concentrations 

Water samples were taken from the control and each test group (replicates Rt -~pooled) at 

0 and 48 hours for quantitative analysis. 0 hour samples were stored at approximately -20 °C 

prior to analysis with the 48 hour samples. 

Duplicate samples were taken and stored at approximately -20 °C for further analysis if 

necessary. 
Harlan Study Number: 41206434 Report Page 13 

Samples at the No Observed Effect Concentration and above only were analyzed. 

The method of analysis, recovery and test preparation analyses are described in Appendix 4. 

3.4.2.5 Evaluation of Data 

The EC5o value and associated confidence limits at 24 hours were calculated by the 

maximum-likelihood probit method (Finney, 1971) and at 48 hours by the trimmed 

Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package 

(ToxCalc, 1999) 

Probit analysis is used where two or more partial responses to exposure are shown. 

When only one partial response is shown the trimmed Spearman-Karber method is appropriate. 
Harlan Study Number: 41206434 Report Page 14 

4 RESULTS 

4.1 Range-finding Test 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the 

range-finding test are given in Table 1. 

No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L. However, 

immobilization was observed at 10 and 100 mg/L. 

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for 

the definitive test. 

Chemical analysis of the 1.0, 10 and 100 mg/L test preparations at 0 hours (see Appendix 4) 

showed measured concentrations of 1.2, 12 and 128 mg/L respectively, and at 48 hours showed 

measured concentrations of 1.2, 12 and 118 mg/L respectively, indicating that the test item was 

stable under test conditions. 

4.2 Definitive Test 

4.2.1 Immobilization Data 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the 

definitive test are given in Table 2. The relationship between percentage immobilization and 

concentration at 24 and 48 hours is given in Figure 1 and Figure 2. 

Analysis ofthe immobilization data by the the probit method (Finney, 1971) at 24 hours and by 

the trimmed Spearman-Karber method (Hamilton et a!, 1977) at 48 hours based on the nominal 

test concentrations gave the following results: 

Time (h) ECso (mg/L) 95% Confidence limits 

(mg/L) 

24 43 29-74 

48 8.5 6.6-11 

The No Observed Effect Concentrations after 24 and 48 hours exposure were 10 and 3.2 mg/L 

respectively. The Lowest Observed Effect Concentration (LOEC) after 24 an4 48 hours 

exposure were considered to be 32 and 10 mg/L respectively. · 

Due to the unsuitable nature of the data it was not possible to calculate the slope and error of 

response curve at 24 or 48 hours. 
Harlan Study Number: 41206434 Report Page 15 

4.2.2 Observations on Test Item Solubility 

The test preparations were observed to be clear colorless solutions for the duration of the test. 

4.2.3 Physico-Chemical Measurements 

The results of the physico-chemical measurements are given in Appendix 5. Temperature was 

maintained at 20 °C to 21 °C throughout the test, while there were no treatment related 

differences for oxygen concentration or pH. 

4.2.4 Verification of Test Concentrations 

Analysis of the test preparations at 0 and 48 hours (see Appendix 4) showed measured test 

concentrations to be near nominal and so it was considered justifiable to calculate the EC50 

values in terms of the nominal test concentrations only. 
Harlan Study Number: 41206434 Report Page 16 

5 CONCLUSION 

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been 

investigated and gave a 48-Hour EC5o value of 8.5 mg/L with 95% confidence limits of 

6.6 - 11 mg/L. The Lowest Observed Effect Concentration was considered to be 10 mg/L. The 

No Observed Effect Concentration at 48 hours was 3.2 mg/L. 
Harlan Study Number: 41206434 Report Page 17 

6 REFERENCES 

FINNEY, D J (1971) Statistical Method in Biological Assay. London : Griffin and Company 

Ltd. 

HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber 

Method for Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol 

11 ' 714-719. 

ToxCalc Version 5.0.23C (1999), Tidepool Scientific Software, McKinleyville, CA 95519, USA. 
Harlan Study Number: 41206434 Report Page 18 

TABLES 

Table 1 Cumulative Immobilization Data in the Range-finding Test 

Nominal Cumulative Immobilized Daphnia 

Concentration (Initial Population: 10 Per Replicate) 

{mg/L) 24 Hours 48 Hours 

Control 0 0 

0.10 0 0 

1.0 0 0 

10 4 4 

100 8 10 
Harlan Study Number: 41206434 Report Page 19 

Table 2 Cumulative Immobilization Data in the Definitive Test 

Cumulative Immobilized Daphnia 

Nominal (Initial Population: 5 Per Replicate) 

Concentration 

(mg/L) 

24 Hours 48 Hours 

Rt R2 RJ ~ Total % Rt R2 R3 R4 Total % 

Control 0 0 0 0 0 0 0 0 0 0 0 0 

1.0 0 0 0 0 0 0 0 0 0 0 0 0 

3.2 0 0 0 0 0 0 0 0 0 0 0 0 

10 0 0 0 2 2 10 1 5 3 4 13 65 

32 4 2 1 3 10 50 5 5 5 5 20 100 

100 4 2 5 3 14 70 5 5 5 5 20 100 

R1 - ~ = Replicates 1 to 4 
Harlan Study Number: 41206434 Report Page 20 

FIGURES 

Figure 1 Concentration-Response Curve after 24 Hours 

99.9 

99 

90 

-~ 

l; 

§< 

Q 

70 

"0 

<I) 

~ 50 

:E 

0 

8 

8 

- 30 

~ 

10 

0.1 

0.01 6-----------o-----'---'-....;_J--------------' 

1 10 100 

Concentration (mg/L) 
Harlan Study Number: 41206434 Report Page 21 

Figure 2 Concentration-Response Curve after 48 Hours 

99.9 

99 

90 

~ 

·;;:: 

.J:: 70 §< 

Q 

"0 

<U 

~ 50 

:.0 

0 

s 

s 

- 30 

'$. 

10 

0.1 

0.01~----------~~----------~----------------~--~--~ 

1 10 100 

Concentration (mg/L) 
Harlan Study Number: 41206434 Report 

APPENDICES 

Appendix 1 Reconstituted Water- Elendt M7 Medium 

Solution 

(I) H3B03 

(II) MnChAH20 

(III) ~iCl 

(IV) RbCl 

(V) SrCh.6H20 

(VI) NaBr 

(VII) Na2Mo04.2H20 

(VIII) CuCh.2H20 

(IX) ZnCh 

(X) CoCh.6H20 

(XI) Kl 

(XII) Na2Se03 

(XIII) NH4V03 

(XIV) Na2EDT A.2H20 

FeS04.7H20 

Concentration of 

stock solution (mg/L) 

57190 

7210 

6120 

1420 

3040 

320 

1260 

335 

260 

200 

65 

43.8 

11.5 

5000 

1991 

Page 22 

An aliquot (dependant on the volume of medium required) of each stock solution was added to a 

final volume of deionized reverse osmosis water to give stock solution A and stored at 

approximately 21 °C. 
Harlan Study Number: 41206434 

Reconstituted Water- Elendt M7 

Macro Nutrient Stock Solutions 

Solution 

(XV) CaCb.2H20 

(XVI) NaHC03 

(XVII) MgS04.1H20 

(XVIII) Na2Si03.9H20 

(XIX) KCl 

(XX) NaN03 

(XXI) K2HP04 

(XXII) KH2P04 

Vitamin Nutrients 

Solution 

(XXIII) Thiamine hydrochloride 

Cyanocobalamine (vitamin B12) 

D(+) biotin (vitamin H) 

Report 

Concentration of 

stock solution (giL) 

293.80 

64.80 

246.60 

50.00 

58.00 

2.74 

1.84 

1.43 

Concentration of 

stock solution (mg/L) 

750 

10 

7.5 

Page 23 

The final medium was prepared by adding an aliquot of stock solution A along with aliquots of 

each individual Macro Nutrient Stock Solution and an aliquot of the vitamin nutrient to the 

required amount (final volume) of deionized reverse osmosis water. 

The pH ofthe prepared media was 8.0 ± 0.2 and stored at approximately 21 °C. 
Harlan Study Number: 41206434 Report Page 24 

Appendix 2 Positive Control 

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the 

reference item at concentrations of0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. 

Exposure conditions for the positive control were similar to those in the definitive test. 

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed 

Spearman-Karber method (Hamilton et a! 1977*) at 48 hours based on the nominal test 

concentrations gave the following results: 

Time (h) ECso (mg/L) 95% Confidence limits 

(mg!L) 

24 0.75 0.56 - 1.0 

48 0.45 0.42 - 0.48 

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L 

respectively. The No Observed Effect Concentration is based upon zero immobilization at this 

concentration. 

The results from the positive control with potassium dichromate were within the normal range 

for this reference item. 

* HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for 

Estimating Median Lethal Concentration in Toxicity Bioassays. Environ Sci Technol11, 714-719. 
Harlan Study Number: 41206434 

Appendix 3 Reconstituted Water 

i) Stock Solutions 

a) 

b) 

c) 

d) 

CaCh.2H20 

MgS04.7H20 

NaHC03 

KCl 

ii) Preparation 

Report 

11.76 g/L 

4.93 g/L 

2.59 giL 

0.23 g/L 

Page 25 

An aliquot (25 mL) of each of solutions a-d was added to each liter (final volume) of 

deionized water with a conductivity of <5 flS cm-1• The reconstituted water had a pH of 

7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved 

oxygen concentration was approximately air-saturation value. 

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as 

CaC03. 
Harlan Study Number: 41206434 Report Page 26 

Appendix 4 Analytical Investigations 

CONTENTS 

CONTENTS .................................................................................................................................. 26 

1 MATERIALS AND METHODS ......................................................................................... 27 

1.1 Introduction ........................................................................................................... 27 

1.2 Test Item ................................................................................................................ 27 

1.3 Analytical Standard ............................................................................................... 27 

1.4 Analytical Procedure ............................................................................................. 27 

1.4.1 Storage ..................................................................................................... 27 

1.4.2 Reagents and Solvents ............................................................................. 27 

1.4.3 Preparation of Calibration Standards ....................................................... 28 

1.4.4 Preparation of Linearity Standards .......................................................... 28 

1.4.5 Preparation of Spiked Recovery Samples ............................................... 28 

1.4.6 Preparation ofTest Samples .................................................................... 28 

1.4. 7 Instrumental Parameters .......................................................................... 28 

1.4.8 Calculations ............................................................................................. 30 

2 RESULTS ............................................................................................................................ 32 

2.1 Range-Finding Test ............................................................................................... 32 

2.2 Definitive Test ....................................................................................................... 32 

2.2.1 Validation of the Analytical Method ....................................................... 32 

2.2.2 Test Samples ............................................................................................ 32 

3 DISCUSSION ...................................................................................................................... 33 

TABLES ........................................................................................................................................ 34 

Table 1 Preparation of Test Samples ................................................................................. 34 

Table 2 Calibration Standard Data .................................................................................... 34 

Table 3 Results for Range-Finding Samples ..................................................................... 35 

Table 4 Results for Spiked Recovery Samples .................................................................. 35 

Table 5 Results for Test Samples ...................................................................................... 36 

FIGURES ...................................................................................................................................... 37 

Figure 1 

Figure 2 

Figure 3 

Figure 4 

Figure 5 

Calibration Plot ...................................................................................................... 37 

Chromatogram of Calibration Solution (1.0 mg/L) ............................................... 38 

Chromatogram of Biological Control Sample ....................................................... 38 

Chromatogram ofTest Sample (3.2 mg/L) ........................................................... 39 

Chromatogram ofTest Sample (100 mg/L) .......................................................... 39 
Harlan Study Number: 41206434 Report Page 27 

Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The test item concentration in the test samples was determined by direct injection to an 

electrospray mass spectrometer (MS) using an external standard. 

The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, 

Shardlow, UK. 

1.2 Test Item 

The test item is described in the biological part of this study. 

1.3 Analytical Standard 

The test item described in the biological part of this study was also used as the analytical 

standard. 

1.4 Analytical Procedure 

1.4.1 Storage 

The 0 hour samples were stored at approximately -20 °C prior to analysis, whilst the 48 hour 

samples were analyzed on the day of receipt. 

1.4.2 Reagents and Solvents 

Water: water, Romil, UpS ultra-gradient grade. 

Test medium: as described in the biological part of this study. 

Solvents: methanol, Fisher Scientific, HPLC grade. 

Reagents: formic acid, Fisher Scientific, analytical reagent grade. 
Harlan Study Number: 41206434 Report Page 28 

Analytical Investigations 

1.4.3 Preparation of Calibration Standards 

The test item (nominal100 mg) was dissolved in (50:50 v/v) methanol:test medium (100 mL) to 

prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further 

diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined 

volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain 

calibration standards in the range of 0.10 to 1.0 mg/L. A second series of calibration standards 

was similarly prepared in the range of 0.30 to 1.5 mg/L. These solutions were used to determine 

the recovery and test sample concentrations. 

1.4.4 Preparation of Linearity Standards 

The test item (nominal100 mg) was dissolved in (50:50 v/v) methanol:test medium (100 mL) to 

prepare a stock solution with a concentration of 1000 mg/L. This stock solution was further 

diluted with (50:50 v/v) methanol:test medium to produce a solution of 10 mg/L. Defined 

volumes of this solution were then diluted with (50:50 v/v) methanol:test medium to obtain 

calibration standards in the range of 0.10 to 1.0 mg/L. A second series of calibration standards 

was similarly prepared in the range of 0.30 to 1.5 mg/L. These standards were used to evaluate 

the linearity of the analytical system. 

1.4.5 Preparation of Spiked Recovery Samples 

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked 

with the test item and its recovery was assessed. The test item (nominal 100 mg) was initially 

dissolved in (50:50 v/v) methanol:test medium to prepare a stock solution with a concentration 

of 1000 mg/L. This stock solution was further diluted with (50:50 v/v) methanol:test medium to 

produce a solution of 100 mg/L. A defined volume of this stock solution was diluted with test 

medium to obtain spiked recovery samples at a concentration of 1.0 mg/L. Five replicates were 

prepared and subjected to the same treatment as the test samples. In addition, test medium 

without the addition of the test item (synthetic control) was also analyzed. 

1.4.6 Preparation of Test Samples 

The 0 hour test samples were thawed with the aid of sonication, whilst the 48 hour samples were 

analyzed on the day of receipt. They were initially diluted with methanol and then further 

diluted into the calibration range with (50: 50 v/v) methanol:test medium as shown in Table 1. 
Harlan Study Number: 41206434 Report 

Analytical Investigations 

1.4. 7 Instrumental Parameters 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Flow rate: 

Injection volume: 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

Fragmentor voltage: 

Drying gas flow: 

Data acquisition mode: 

SIM ion: 

Agilent 1200 Series 

Agilent 1200 Series 

Agilent 1200 Series MSD 

no column 

water:methanol:formic acid (500:500:0.5 v/v/v) 

0.5 mL/minute 

10 ~L 

ESI 

positive 

1000V 

350 oc 

40 psi 

100V 

10 L/min 

Single Ion Monitoring (SIM) 

Page 29 
Harlan Study Number: 41206434 Report Page 30 

Analytical Investigations 

1.4.8 Calculations 

A calibration curve was prepared using a quadratic function of the form shown in equation (1). 

The calibration data is presented in Table 2, and Figure 1. From the calibration curve, the 

concentration "x" of the test item in an injected sample was calculated using the following 

equation: 

-b+~b 2 -4·a·(c- y) X=---=-------'-- (1) 

2·a 

where x: 

y: 

a, b, c: 

concentration ofthe test item in injected sample [mg/L] 

average peak area of the test item in injected sample [counts] 

constants 

The concentration of the test item in the test samples was calculated using the following 

equation: 

c = X ·F 

R/100 

where c: 

x: 

F: 

R: 

(2) 

concentration of the test item in the test sample [ mg/L] 

concentration of the test item in the injected sample solution [mg!L] 

sample preparation factor 

recovery rate[%] (If not correcting for recovery then R is taken as 100) 

The recovery rate (R) of the spiked recovery samples was calculated using the following 

equation: 

R =~·100 cfort 

where R: 

c: 

(3) 

recovery rate [%] 

determined concentration of the test item in the spiked recovery sample 

[mg/L] 

fortified concentration of the test item in the spiked recovery sample 

[mg!L] 
Harlan Study Number: 41206434 Report Page 31 

Analytical Investigations 

The percentage of nominal concentration of the test item in the test samples was calculated using 

the following equation: 

%nominal= _c_ ·1 00 

cnom 

(4) 

where 

Cnom: nominal concentration in the test sample [mg/L] 
Harlan Study Number: 41206434 Report Page 32 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

The analytical methodology was not validated prior to the analysis of range-finding samples; full 

validation was later conducted specifically over concentration range to be used in the definitive 

test. The results for the range-finding test samples are summarized in Table 3. 

2.2 Definitive Test 

2.2.1 Validation of the Analytical Method 

Linearity 

The calibration data for the calibration standards of the test item is given in Table 2, and Figure 1. 

The data was found to have a quadratic correlation within the calibration range of 

0 to 1.84 mg/L. The R2 fit of the calibration curve to the data was 0.9997, and was considered to 

be acceptable. 

Accuracy (Recovery) and Precision 

A set of recovery samples accurately fortified at a relevant concentration of test item was 

prepared five-fold and analyzed. The results obtained for the concentration of the test item in the 

recovery samples are presented in Table 4. 

The method was considered to be sufficiently accurate and precise for the purposes of this test. 

The test sample results were not corrected for recovery. 

Limit of Quantitation (LOQ) 

The limit of quantitation (LOQ) was determined by calculating the concentration equivalent to 

ten times the standard deviation of five synthetic controls. Using this method the limit of 

quantitation was determined to be 0.053 mg/L. 

2.2.2 Test Samples 

The results obtained for the concentration of the test item found in the test samples aie presented 

in Table 5. 

Typical chromatograms are shown in Figures 2 to 5. 
Harlan Study Number: 41206434 Report Page 33 

Analytical Investigations 

3 DISCUSSION 

The detection system was found to have acceptable correlation to the fit of a quadratic equation. 

The analytical procedure was found to have acceptable recoveries of test item in test medium. 

The method of analysis was validated and proven to be suitable for use. 
Harlan Study Number: 41206434 Report Page 34 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Nominal Sample Sample Final Nominal 

Concentration of Volume Final Volume Preparation Concentration of Test 

Test Item Factor Item 

[mg/L] [mL] [mL] F [mg/L] 

Control 5 10 2 -

3.2 5 10, then 4 0.80 5--710 

10 5 10, then 10 1.0 4--720 

32 5 10, then 40 0.80 1--720 

100 5 10, then 100 1.0 2--7100 

not applicable 

Table 2 Calibration Standard Data 

Concentration of Test Item Area 

[mg/L] [counts] 

0 2.245 X 104 

0.0950 3.166 X 104 

0.368 5.370 X 104 

0.475 6.281 X 104 

0.858 9.225 X 104 

0.950 9.711 X 104 

1.838 1.607 X 105 
Harlan Study Number: 41206434 Report Page 35 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Nominal Measured Sample Determined Concentration 

of Concentration Preparation Concentration 

Test Item in of Test Item in Factor of Test Item in %of Nominal Time Point Range-Finding Sample Vial Range-Finding Concentration 

Sample Sample 

X F c Cnom 

[hours] [mg!L] [mg/L] [mg/L] [%] 

0 1.0 0.595 2 1.19 119 

10 1.17 10 11.7 117 

100 1.28 100 128 128 

48 1.0 0.595 2 1.19 119 

10 1.20 10 12.0 120 

100 1.18 100 118 118 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Analytical 

Concentration Concentration Concentration Preparation Concentration Recovery Precision 

of of Test Item in of Test Item in Factor of Test Item in Rate (Relative 

Test Item the Spiked the Sample the Spiked Standard 

Sample Vial Sample Deviation of 

Recovery) 

Cnom Crort X F c R 

[mg/L] [mg/L] [mg/L] [mg!L] [%] [%] 

1.0 0.948 0.362 2 0.724 

0.406 2 0.811 

0.397 2 0.794 80 5.8 . 0.354 2 0.707 

0.378 2 0.756 

Acceptance Target 80-120 <10 

LOQ Limit of Quantitation 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41206434 Report 

Analytical Investigations 

Table 5 Results for Test Samples 

Nominal Measured Sample 

Concentration Concentration Preparation 

Time Point 

[hours] 

0 

48 

LOQ 

of of Test Item in 

Test Item in Sample Vial 

Test Sample 

Cnom X 

[mg/L] [mg/L] 

Control <LOQ 

3.2 0.770 

10 0.945 

32 0.770 

100 1.00 

Control <LOQ 

3.2 0.735 

10 0.958 

32 0.793 

100 0.962 

Limit of Quantitation 

not applicable 

For reporting purposes, x has been calculated from c retrospectively. 

Factor 

F 

2 

4 

10 

40 

100 

2 

4 

10 

40 

100 

Page 36 

Determined 

Concentration 

of Test Item in %of Nominal Test Sample Concentration 

c 

[mg/L] [%] 

<LOQ - 3.08 96 

9.45 95 

30.8 96 

100 100 

<LOQ - 2.94 92 

9.58 96 

31.7 99 

96.2 96 
Harlan Study Number: 41206434 

Analytical Investigations 

FIGURES 

Figure 1 Calibration Plot 

2 180000 

·;:: 

2 160000 co 

I!! 

<( 140000 

-"" co 

(J) 

a.. 120000 

100000 

80000 

60000 

40000 

20000 

0 

0.00 

Report 

y = -5.106E+03x2 + 8.408E+04x + 2.326E+04 

R2 = 9.997E-01 

0.50 1.00 

Page 37 

1.50 2.00 

Concentration (mg/L) 
Harlan Study Number: 41206434 Report Page 38 

Analytical Investigations 

Figure 2 Chromatogram of Calibration Solution (1.0 mg!L) 

Figure 3 Chromatogram of Biological Control Sample* 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min 

• Background interference level of the response. Not test item. 
Harlan Study Number: 41206434 Report Page 39 

Analytical Investigations 

Figure 4 Chromatogram of Test Sample (3.2 mg/L) 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min 

Figure 5 Chromatogram of Test Sample (100 mg/L) 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 min 
Harlan Study Number: 41206434 Report 

Appendix 5 Physico-Chemical Measurements 

Nominal 0 Hours 24 Hours 48 Hours 

Concentration 

(mg/L) pH mg %ASV* T°C T"C pH mg 0 2/L 0 2/L 

RI 8.0 9.1 100 20 21 8.1 8.8 

Control Rz 8.0 9.0 99 20 21 8.1 8.7 

R3 8.0 9.0 99 20 21 8.1 8.7 

~ 8.0 8.9 98 20 21 8.1 8.6 

R1 8.0 8.9 98 20 21 8.1 8.7 

1.0 Rz 8.0 8.9 98 20 21 8.1 8.6 

R3 8.0 8.9 98 20 21 8.1 8.6 

~ 8.0 8.9 98 20 21 8.1 8.6 

R1 8.0 8.9 98 20 21 8.1 8.6 

3.2 Rz 8.0 8.9 98 20 21 8.1 8.6 

R3 8.0 8.9 98 20 21 8.1 8.6 

~ 8.0 8.9 98 20 21 8.0 8.6 

R1 8.0 8.9 98 20 21 8.0 8.7 

10 Rz 8.0 8.9 98 20 21 8.0 8.7 

R3 8.0 8.9 98 20 21 8.0 8.7 

~ 8.0 8.9 98 20 21 8.0 8.7 

RI 8.0 8.9 98 20 21 8.0 8.8 

32 Rz 7.9 8.9 98 20 21 8.0 8.7 

R3 7.9 8.9 98 20 21 8.0 8.7 

~ 7.9 8.9 98 20 21 8.0 8.7 

RI 7.9 8.9 98 20 21 8.0 8.7 

100 Rz 7.8 8.9 98 20 21 8.0 8.7 

R3 7.8 8.9 98 20 21 8.0 8.7 

~ 7.8 8.9 98 20 21 8.0 8.7 

* ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value 

R1 - Rt = Replicates 1 to 4 

%ASV* 

99 

98 

98 

97 

98 

97 

97 

97 

97 

97 

97 

97 

98 

98 

98 

98 

99 

98 

98 

98 

98 

98 

98 

98 

Page 40 

T°C 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 

21 
Harlan Study Number: 41206434 Report 

Appendix 6 Monitoring Authority Statement of GLP Compliance 

Department 

of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY 

Harlan Laboratories Ltd 

Shardlow Business Park 

London Road 

Shardlow 

Derby 

m:722GD 

DATE OF INSPECTION 

10 July 2012 

TEST TYPE(S) 

Analytical/Clinical 

Chemistry 

Environmental Toxicity 

Environmental Fate 

Mutagenicity 

Phys/Chem. Tests 

Toxicology 

An inspection for compliance with the Principles of Good Laboratory Practice was 

carried out at the above test facility as part of the UK Good Laboratory Practice 

Compliance Monitoring Programme. 

This statement confirms that, on the date of issue, the UK Good 

Practice Monitoring Authority were satisfied that the above test facility was 

operating in compliance with the OECD Principles of Good Laboratory Practice. 

This statement constitutes a Good Laboratory Practice Instrument (as defined in the 

UK Good Laboratory Practice Regulations 1999). 

Dr. Andrew J. Gray 

Head, UK GLP Monitoring Authority 

Page 41